Abstract: Major causes of vision loss in people with diabetes include diabetic macular edema and severe diabetic retinopathy (including severe non-proliferative diabetic retinopathy and proliferative diabetic retinopathy), both of which are associated with extensive injury of retinal endothelial cells; however, the molecular mechanisms underlying the progressive damage to endothelial cells in diabetes are poorly understood. Further, despite preventable measures, severe diabetic retinopathy still occurs in a large number of diabetic individuals eventually leading to blindness. Thus, identifying novel molecular targets to prevent the progression of diabetic retinopathy is an unmet need. In this project, we propose to test the role of NADPH oxidase 4 (Nox4) in retinal endothelial cells as a novel molecular target for the prevention and treatment of severe diabetic retinopathy. In recent studies, we have shown that Nox4 is a major isoform of NADPH oxidases in retinal endothelial cells and Nox4 expression is upregulated in retinal blood vessels in animal models of diabetic retinopathy. Genetic ablation of Nox4 gene in endothelial cells reduces retinal vascular permeability in diabetic mice and alleviates aberrant new vessel growth in oxygen-induced retinopathy. In contrast, overexpression of Nox4 in endothelial cells is sufficient to induce retinal vascular leakage and degeneration. Furthermore, a recent genome-wide association study (GWAS) identified Nox4 as a potential risk gene for severe diabetic retinopathy. Collectively, these experimental and clinical findings suggest an important role of Nox4 in diabetes-mediated endothelial cell injury and retinal vasculopathy. However, the mechanisms by which Nox4 induces endothelial injury and its relevance to human disease remain an understudied area. In this project, we will first characterize Nox4 expression in human retinas with different stages of diabetic retinopathy. Using an inducible endothelial cell specific conditional knockout mouse line, we will delete Nox4 at various time points after diabetes onset to establish the role of endothelial Nox4 in the development and progression of diabetic retinopathy. We will investigate the mechanisms of Nox4- mediated endothelial death and senescence and elucidate novel signaling pathways activated by Nox4 upregulation in endothelial cells. Finally, we will test novel pharmacological inhibitor that specifically targets Nox4 for its preventive and therapeutic effects on diabetic retinopathy. We anticipate that the successful completion of the proposed studies will not only provide mechanistic insight of diabetic retinopathy but also develop new treatment that targets a risk gene identified by human genetic study for prevention of severe diabetic retinopathy.